The principal goal of this research is to understand telomerase regulation. Specifically, it will focus on elucidating the mechanism of how telomerase preferentiallyelongates short telomeres. A telomere is a complex of DNAand proteins that marks the end of a chromosome. Telomerase is an enzymethat elongates telomeres, thereby enabling cells to continuouslydivide without losingtheir genetic material. In both lower and higher eukaryotes, telomerase preferentiallyelongates short telomeres. Previous findings from our lab show that in yeast the protein content of short telomeres is different from that of WT-length telomeres. We speculate that this difference marks short telomeres for telomerase recruitment. Rifip and Rif2p, known negative regulators of telomerase, were initially predicted to be lost from short telomeres, thereby enhancing telomerase activity. Surprisingly,in these previous studies, Rifip bound equallywell to short and long telomeres while Rif2p binding was decreased. In contrast, Telip showed preferentialbindingfor short telomeres. Telip is an ATM/ATR-like kinase, shown to be necessary for recruiting telomerase subunits to short telomeres. A hypothesis consistent with these findings is that the reduced Rif2p binding at short telomeres recruits Telip, to that telomere, Telip phosphorylates Rifip, and this phosphorylation relieves its ability to inhibit telomerase in cis. Accordingto this model, the inactivation, not the displacement, of Rifip, allows telomerase recruitment and activation. This proposal will test these ideas with the following aims: (i) confirm the reduced Rif2p and WT-level Rifip binding to short telomeres using telomeres shortened from their ends as occurs in vivo, (2) determine if Rifip is regulated by Telip-dependent phosphorylation, and (3) assess if Rifip phosphorylation affects telomerase action. For aim i, a yeast strain, deleted in the telomerase catalytic subunit, will be used to shorten telomeres. Using this strain, chromatin IP and quantitativePCR will be performed to assess the binding levels of Rifip and Rif2p on short telomeres. For aim 2, a synthetic dosage lethality screen will be employed to evaluate whether or not Rifip is a phosphorylation target of Telip. For aim 3, site-directed mutagenesis of Telip phosphorylation consensus sites will be performed to identify sites of Telip action on Rifip. Subsequently, telomere lengths will be assessed to seeif phosphorylation of these residues affects the ability of telomerase to lengthen telomeres. As a large portion ofthe U.S. population reaches their retirement age, age-related diseases arebecoming more prevalent. Therefore, it is important to understand how cells ageby studying telomerase regulation. Once the clear mechanism is understood, it can be used to develop a new therapy to slow cell aging, the most fundamental problem in all age-related diseases, thereby serving the needs of public health at large.